HOXA9 methylation detection by droplet digital PCR and blood vessel invasion using FFPE tissues for prognostic stratification of stage I lung adenocarcinoma patients

Abstract Surgical resection is the only recommended curative treatment for stage I non-small cell lung cancer (NSCLC) patients, as the benefit of adjuvant chemotherapy remains controversial. After complete resection patients with disease at the same stage experience different outcomes, and within 5 years a third of patients relapse. With the implementation of low-dose computed tomography scan for lung cancer screening, the number of lung cancer patients diagnosed at stage I is projected to rise. Therefore, prognostic biomarkers are urgently needed to more accurately predict recurrence following surgery and potentially guide the decision to administer adjuvant chemotherapy for high-risk patients. In the present study, we evaluated the prognostic significance of two biomarkers, namely HOXA9 promoter methylation and blood vessel invasion (BVI), for risk stratification of stage I lung adenocarcinoma patients. The type of biospecimens and the choice of assay platform are key issues to foster translation of biomarkers to the clinic. Here, we demonstrated the application of a droplet digital PCR (ddPCR)-based assay to analyze HOXA9 promoter methylation in formalin-fixed, paraffin-embedded (FFPE) tumor specimens, generated during routine pathologic assessment of resected patients. In recent years, ddPCR has become increasingly used clinically due to its ability to reliably detect and quantitate rare alleles, as well as its technical simplicity, rapidity and cost effectiveness. We replicated previous observations that HOXA9 promoter is methylated de novo in stage I tumors (P <0.0001). Using 177 FFPE tumor samples, we showed that high methylation was associated with worse cancer-specific survival (Hazard Ratio [HR], 3.37; P=0.0002) and identified high-risk stage IA and IB patients. Importantly, addition of this molecular marker improved a risk model comprising clinical and pathologic parameters (Nested likelihood ratio test; P=0.003). HOXA9 promoter methylation was associated with a transcriptome signature enriched in genes marked by Polycomb in Embryonic Stem Cells, a signature previously associated with poor differentiation and worse overall patient survival. Moreover, BVI was independently associated with poor outcome (HR, 2.62; P=0.054). A score that combined BVI with HOXA9 promoter methylation further stratified high-risk patients (Trend P=0.0001 comparing 0, 1 or 2 positive markers). Collectively, our results support the use of ddPCR to quantify HOXA9 promoter methylation and BVI determination from routine pathology FFPE specimens, to identify patients at high risk of recurrence. If validated in a larger independent study, our findings could help inform patient management in prospective clinical trials that evaluate the benefit of adjuvant chemotherapy in early-stage invasive lung adenocarcinoma to prevent recurrence. Citation Format: Delphine Lissa, Teruhide Ishigame, Rintaro Noro, Marguerite J. Tucker, Valery Bliskovsky, Steven Shema, Elise D. Bowman, Curtis C. Harris, Ana I. Robles. HOXA9 methylation detection by droplet digital PCR and blood vessel invasion using FFPE tissues for prognostic stratification of stage I lung adenocarcinoma patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4208.